Amino compounds are abundant within the renal inner medulla, but their possible role during hypertonic stress is not clear. Renal epithelial Madin-Darby canine kidney cells were used to examine the osmoregulation of system A transport, a major Na(+)-dependent process for neutral amino acid transport. System A activity was markedly increased after 6 h of hypertonic challenge, and intracellular alanine content increased more than twofold. The activation of system A was reversed after 24 h of hypertonic challenge. This downregulation was accompanied by the activation of betaine transport, as measured by gamma-aminobutyric acid uptake. Extracellular betaine prevented the early activation of system A. The hypertonic activation of system A was blocked by actinomycin D and cycloheximide. When cells were returned to isotonic medium after hypertonic activation, the recovery of system A transport also was partially inhibited by actinomycin D and puromycin. The results are consistent with the possibility that hypertonicity, by disrupting a repressor protein, leads to increased synthesis of a system A-related protein. The isotonic recovery may require synthesis of new repressor proteins.